PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955

Figure 1. The relationship between the protein concentration of lysates from untreated and H2O2-treated C2C12 cells and the absorbance at 450 nm using the PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955 is shown.

Figure 1. The relationship between the protein concentration of lysates from untreated and H2O2-treated C2C12 cells and the absorbance at 450 nm using the PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955 is shown.

Figure 1. The relationship between the protein concentration of lysates from untreated and H2O2-treated C2C12 cells and the absorbance at 450 nm using the PathScan® Phospho-AMPKα (Thr172) Sandwich ELISA Antibody Pair #7955 is shown.

Wash four times with wash buffer, 200 μl each time per well. For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.

D. Test Procedure

Lysates can be used undiluted or diluted in blocking buffer. 100 μl of lysate is added per well. Cover plate and incubate at 37°C for 2 hr.

Wash plate (Section C, Step 3).

Dilute detection antibody 1:100 in blocking buffer. For a single 96 well plate, add 100 μl of detection antibody Stock to 9.9 ml of blocking buffer. Mix well and add 100 μl/well. Cover plate and incubate at 37°C for 1 hr.

Wash plate (Section C, Step 3).

Secondary antibody, either streptavidin anti-mouse or anti-rabbit-HRP, is diluted 1:1000 in blocking buffer. For a single 96 well plate, add 10 μl of secondary antibody stock to 9.99 ml of blocking buffer. Mix well and add 100 μl/well. Cover and incubate at 37°C for 30 min.

Wash plate (Section C, Step 3).

Add 100 μl of TMB substrate per well. Cover and incubate at 37°C for 10 min.

Storage:

For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

AMP-activated protein kinase (AMPK) is highly conserved from yeast to plants and animals and plays a key role in the regulation of energy homeostasis (1). AMPK is a heterotrimeric complex composed of a catalytic α subunit and regulatory β and γ subunits, each of which is encoded by two or three distinct genes (α1, 2; β1, 2; γ1, 2, 3) (2). The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress, such as heat shock, hypoxia, and ischemia (1). The tumor suppressor LKB1, in association with accessory proteins STRAD and MO25, phosphorylates AMPKα at Thr172 in the activation loop, and this phosphorylation is required for AMPK activation (3-5). AMPKα is also phosphorylated at Thr258 and Ser485 (for α1; Ser491 for α2). The upstream kinase and the biological significance of these phosphorylation events have yet to be elucidated (6). The β1 subunit is post-translationally modified by myristoylation and multi-site phosphorylation including Ser24/25, Ser96, Ser101, Ser108, and Ser182 (6,7). Phosphorylation at Ser108 of the β1 subunit seems to be required for the activation of AMPK enzyme, while phosphorylation at Ser24/25 and Ser182 affects AMPK localization (7). Several mutations in AMPKγ subunits have been identified, most of which are located in the putative AMP/ATP binding sites (CBS or Bateman domains). Mutations at these sites lead to reduction of AMPK activity and cause glycogen accumulation in heart or skeletal muscle (1,2). Accumulating evidence indicates that AMPK not only regulates the metabolism of fatty acids and glycogen, but also modulates protein synthesis and cell growth through EF2 and TSC2/mTOR pathways, as well as blood flow via eNOS/nNOS (1).

Upstream / Downstream

Explore pathways related to this product.

Upstream / Downstream Proteins

STRING - Known and Predicted Protein-Protein Interactions.

Important Ordering Details

Custom Ordering Details:
Product is assembled upon order to ensure maximum activity. United States: Please allow up to two weeks for your order to be processed and shipped. Outside of the United States: Please allow up to three weeks, depending on the country, for your order to be processed and shipped. When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.